The NuRD complex and macrocephaly associated neurodevelopmental disorders

The nucleosome remodeling and deacetylase (NuRD) complex is a major regulator of gene expression involved in pluripotency, lineage commitment, and corticogenesis. This important complex is composed of seven different proteins, with mutations in CHD3, CHD4, and GATAD2B being associated with neurodevelopmental disorders presenting with macrocephaly and intellectual disability similar to other overgrowth and intellectual disability (OGID) syndromes. Pathogenic variants in CHD3 and CHD4 primarily involve disruption of enzymatic function. GATAD2B variants include loss‐of‐function mutations that alter protein dosage and missense variants that involve either of two conserved domains (CR1 and CR2) known to interact with other NuRD proteins. In addition to macrocephaly and intellectual disability, CHD3 variants are associated with inguinal hernias and apraxia of speech; whereas CHD4 variants are associated with skeletal anomalies, deafness, and cardiac defects. GATAD2B‐associated neurodevelopmental disorder (GAND) has phenotypic overlap with both of these disorders. Of note, structural models of NuRD indicate that CHD3 and CHD4 require direct contact with the GATAD2B‐CR2 domain to interact with the rest of the complex. Therefore, the phenotypic overlaps of CHD3‐ and CHD4‐related disorders with GAND are consistent with a loss in the ability of GATAD2B to recruit CHD3 or CHD4 to the complex. The shared features of these neurodevelopmental disorders may represent a new class of OGID syndrome: the NuRDopathies.     

Variants in PRKAR1B cause a neurodevelopmental disorder with autism spectrum disorder,apraxia, and insensitivity to pain

PurposeWe characterize the clinical and molecular phenotypes of six unrelated individuals with intellectual disability and autism spectrum disorder who carry heterozygous missense variants of the PRKAR1B gene, which encodes the R1β subunit of the cyclic AMP-dependent protein kinase A (PKA).MethodsVariants of PRKAR1B were identified by single- or trio-exome analysis. We contacted the families and physicians of the six individuals to collect phenotypic information, performed in vitro analyses of the identified PRKAR1B-variants, and investigated PRKAR1B expression during embryonic development.ResultsRecent studies of large patient cohorts with neurodevelopmental disorders found significant enrichment of de novo missense variants in PRKAR1B. In our cohort, de novo origin of the PRKAR1B variants could be confirmed in five of six individuals, and four carried the same heterozygous de novo variant c.1003C>T (p.Arg335Trp; NM_001164760). Global developmental delay, autism spectrum disorder, and apraxia/dyspraxia have been reported in all six, and reduced pain sensitivity was found in three individuals carrying the c.1003C>T variant. PRKAR1B expression in the brain was demonstrated during human embryonal development. Additionally, in vitro analyses revealed altered basal PKA activity in cells transfected with variant-harboring PRKAR1B expression constructs.ConclusionOur study provides strong evidence for a PRKAR1B-related neurodevelopmental disorder.     

KAT6A Syndrome: genotype–phenotype correlation in 76 patients with pathogenic KAT6A variants

PurposePathogenic variants in KAT6Ahave recently been identified as a cause of syndromic developmental delay. Within 2 years, the number of patients identified with pathogenic KAT6A variants has rapidly expanded and the full extent and variability of the clinical phenotype has not been reported.MethodsWe obtained data for patients with KAT6A pathogenic variants through three sources: treating clinicians, an online family survey distributed through social media, and a literature review.ResultsWe identified 52 unreported cases, bringing the total number of published cases to 76. Our results expand the genotypic spectrum of pathogenic variants to include missense and splicing mutations. We functionally validated a pathogenic splice-site variant and identified a likely hotspot location forde novo missense variants. The majority of clinical features in KAT6A syndrome have highly variable penetrance. For core features such as intellectual disability, speech delay, microcephaly, cardiac anomalies, and gastrointestinal complications, genotype– phenotype correlations show that late-truncating pathogenic variants (exons 16–17) are significantly more prevalent. We highlight novel associations, including an increased risk of gastrointestinal obstruction.ConclusionOur data expand the genotypic and phenotypic spectrum for individuals with genetic pathogenic variants in KAT6A and we outline appropriate clinical management.     

Refining the Primrose syndrome phenotype: A study of five patients with ZBTB20 de novo variants and a review of the literature

Primrose syndrome is a rare autosomal dominant condition caused by heterozygous missense variants within ZBTB20. Through an exome sequencing approach (as part of the Deciphering Developmental Disorders [DDD] study) we have identified five unrelated individuals with previously unreported, de novo ZBTB20 pathogenic missense variants. All five missense variants targeted the C2H2 zinc finger domains. This genotype‐up approach has allowed further refinement of the Primrose syndrome phenotype. Major characteristics (>90% individuals) include an intellectual disability (most frequently in the moderate range), a recognizable facial appearance and brain MRI abnormalities, particularly abnormalities of the corpus callosum. Other frequent clinical associations (in 50–90% individuals) include sensorineural hearing loss (83%), hypotonia (78%), cryptorchidism in males (75%), macrocephaly (72%), behavioral issues (56%), and dysplastic/hypoplastic nails (57%). Based upon these clinical data we discuss our current management of patients with Primrose syndrome.     

Variant recurrence in neurodevelopmental disorders: the use of publicly available genomic data identifies clinically relevant pathogenic missense variants

PurposeNext-generation sequencing has revealed the major impact of de novo variants (DNVs) in developmental disorders (DD) such as intellectual disability, autism, and epilepsy. However, a substantial fraction of these predicted pathogenic DNVs remains challenging to distinguish from background DNVs, notably the missense variants acting via nonhaploinsufficient mechanisms on specific amino acid residues. We hypothesized that the detection of the same missense variation in at least two unrelated individuals presenting with a similar phenotype could be a powerful approach to reveal novel pathogenic variants.MethodsWe looked for variations independently present in both our database of >1200 solo exomes and in denovo-db, a large, publicly available collection of de novo variants identified in patients with DD.ResultsThis approach identified 30 variants with strong evidence of pathogenicity, including variants already classified as pathogenic or probably pathogenic by our team, and also several new variants of interest in known OMIM genes or in novel genes. We identified FEM1B and GNAI2 as good candidate genes for syndromic intellectual disability and confirmed the implication of ACTL6B in a neurodevelopmental disorder.ConclusionAnnotation of local variants with denovo-db can highlight missense variants with high potential for pathogenicity, both facilitating the time-consuming reanalysis process and allowing novel DD gene discoveries.     

Clinical and molecular consequences of disease-associated de novo mutations in SATB2

PurposeTo characterize features associated with de novo mutations affecting SATB2 function in individuals ascertained on the basis of intellectual disability.MethodsTwenty previously unreported individuals with 19 different SATB2 mutations (11 loss-of-function and 8 missense variants) were studied. Fibroblasts were used to measure mutant protein production. Subcellular localization and mobility of wild-type and mutant SATB2 were assessed using fluorescently tagged protein.ResultsRecurrent clinical features included neurodevelopmental impairment (19/19), absent/near absent speech (16/19), normal somatic growth (17/19), cleft palate (9/19), drooling (12/19), and dental anomalies (8/19). Six of eight missense variants clustered in the first CUT domain. Sibling recurrence due to gonadal mosaicism was seen in one family. A nonsense mutation in the last exon resulted in production of a truncated protein retaining all three DNA-binding domains. SATB2 nuclear mobility was mutation-dependent; p.Arg389Cys in CUT1 increased mobility and both p.Gly515Ser in CUT2 and p.Gln566Lys between CUT2 and HOX reduced mobility. The clinical features in individuals with missense variants were indistinguishable from those with loss of function.ConclusionSATB2 haploinsufficiency is a common cause of syndromic intellectual disability. When mutant SATB2 protein is produced, the protein appears functionally inactive with a disrupted pattern of chromatin or matrix association.Genet Med advance online publication 02 February 2017     

The CHD8 overgrowth syndrome: A detailed evaluation of an emerging overgrowth phenotype in 27 patients

CHD8 has been reported as an autism susceptibility/intellectual disability gene but emerging evidence suggests that it additionally causes an overgrowth phenotype. This study reports 27 unrelated patients with pathogenic or likely pathogenic CHD8 variants (25 null variants, two missense variants) and a male:female ratio of 21:6 (3.5:1, p < .01). All patients presented with intellectual disability, with 85% in the mild or moderate range, and 85% had a height and/or head circumference ≥2 standard deviations above the mean, meeting our clinical criteria for overgrowth. Behavioral problems were reported in the majority of patients (78%), with over half (56%) either formally diagnosed with an autistic spectrum disorder or described as having autistic traits. Additional clinical features included neonatal hypotonia (33%), and less frequently seizures, pes planus, scoliosis, fifth finger clinodactyly, umbilical hernia, and glabellar hemangioma (≤15% each). These results suggest that, in addition to its established link with autism and intellectual disability, CHD8 causes an overgrowth phenotype, and should be considered in the differential diagnosis of patients presenting with increased height and/or head circumference in association with intellectual disability.     

Delineation of a KDM2B-related neurodevelopmental disorder and its associated DNA methylation signature

PurposePathogenic variants in genes involved in the epigenetic machinery are an emerging cause of neurodevelopment disorders (NDDs). Lysine-demethylase 2B (KDM2B) encodes an epigenetic regulator and mouse models suggest an important role during development. We set out to determine whether KDM2B variants are associated with NDD.MethodsThrough international collaborations, we collected data on individuals with heterozygous KDM2B variants. We applied methylation arrays on peripheral blood DNA samples to determine a KDM2B associated epigenetic signature.ResultsWe recruited a total of 27 individuals with heterozygous variants in KDM2B. We present evidence, including a shared epigenetic signature, to support a pathogenic classification of 15 KDM2B variants and identify the CxxC domain as a mutational hotspot. Both loss-of-function and CxxC-domain missense variants present with a specific subepisignature. Moreover, the KDM2B episignature was identified in the context of a dual molecular diagnosis in multiple individuals. Our efforts resulted in a cohort of 21 individuals with heterozygous (likely) pathogenic variants. Individuals in this cohort present with developmental delay and/or intellectual disability; autism; attention deficit disorder/attention deficit hyperactivity disorder; congenital organ anomalies mainly of the heart, eyes, and urogenital system; and subtle facial dysmorphism.ConclusionPathogenic heterozygous variants in KDM2B are associated with NDD and a specific epigenetic signature detectable in peripheral blood.     

Expansion of clinical and variant spectrum of EEF2-related neurodevelopmental disorder: Report of two additional cases

Eukaryotic translation elongation factor 2 (eEF2), encoded by the gene EEF2, is an essential factor involved in the elongation phase of protein translation. A specific heterozygous missense variant (p.P596H) in EEF2 was originally identified in association with autosomal dominant adult-onset spinocerebellar ataxia-26 (SCA26). More recently, additional heterozygous missense variants in this gene have been described to cause a novel, childhood-onset neurodevelopmental disorder with benign external hydrocephalus. Herein, we report two unrelated individuals with a similar gene-disease correlation to support this latter observation. Patient 1 is a 7-year-old male with a previously reported, de novo missense variant (p.V28M) who has motor and speech delay, autism spectrum disorder, failure to thrive with relative macrocephaly, unilateral microphthalmia with coloboma and eczema. Patient 2 is a 4-year-old female with a novel de novo nonsense variant (p.Q145X) with motor and speech delay, hypotonia, macrocephaly with benign ventricular enlargement, and keratosis pilaris. These additional cases help to further expand the genotypic and phenotypic spectrum of this newly described EEF2-related neurodevelopmental syndrome.     

Functional characterization of 84 PALB2 variants of uncertain significance

PurposeInherited pathogenic variants in PALB2 are associated with increased risk of breast and pancreatic cancer. However, the functional and clinical relevance of many missense variants of uncertain significance (VUS) identified through clinical genetic testing is unclear. The ability of patient-derived germline missense VUS to disrupt PALB2 function was assessed to identify variants with potential clinical relevance.MethodsThe influence of 84 VUS on PALB2 function was evaluated using a cellular homology directed DNA repair (HDR) assay and VUS impacting activity were further characterized using secondary functional assays.ResultsFour (~5%) variants (p.L24S,c.71T>C; p.L35P,c.104T>C; pI944N,c.2831T>A; and p.L1070P,c.3209T>C) disrupted PALB2-mediated HDR activity. These variants conferred sensitivity to cisplatin and a poly(ADP-ribose) polymerase (PARP) inhibitor and reduced RAD51 foci formation in response to DNA damage. The p.L24S and p.L35P variants disrupted BRCA1–PALB2 protein complexes, p.I944N was associated with protein instability, and both p.I944N and p.L1070P mislocalized PALB2 to the cytoplasm.ConclusionThese findings show that the HDR assay is an effective method for screening the influence of inherited variants on PALB2 function, that four missense variants impact PALB2 function and may influence cancer risk and response to therapy, and suggest that few inherited PALB2 missense variants disrupt PALB2 function in DNA repair.     

The pleiotropy associated with de novo variants in CHD4, CNOT3, and SETD5 extends to moyamoya angiopathy

PurposeMoyamoya angiopathy (MMA) is a cerebrovascular disease characterized by occlusion of large arteries, which leads to strokes starting in childhood. Twelve altered genes predispose to MMA but the majority of cases of European descent do not have an identified genetic trigger.MethodsExome sequencing from 39 trios were analyzed.ResultsWe identified four de novo variants in three genes not previously associated with MMA: CHD4, CNOT3, and SETD5. Identification of additional rare variants in these genes in 158 unrelated MMA probands provided further support that rare pathogenic variants in CHD4 and CNOT3 predispose to MMA. Previous studies identified de novo variants in these genes in children with developmental disorders (DD), intellectual disability, and congenital heart disease.ConclusionThese genes encode proteins involved in chromatin remodeling, and taken together with previously reported genes leading to MMA-like cerebrovascular occlusive disease (YY1AP1, SMARCAL1), implicate disrupted chromatin remodeling as a molecular pathway predisposing to early onset, large artery occlusive cerebrovascular disease. Furthermore, these data expand the spectrum of phenotypic pleiotropy due to alterations of CHD4, CNOT3, and SETD5 beyond DD to later onset disease in the cerebrovascular arteries and emphasize the need to assess clinical complications into adulthood for genes associated with DD.     

The CHD4-related syndrome: a comprehensive investigation of the clinical spectrum,genotype–phenotype correlations,and molecular basis

PurposeSifrim–Hitz–Weiss syndrome (SIHIWES) is a recently described multisystemic neurodevelopmental disorder caused by de novo variants inCHD4. In this study, we investigated the clinical spectrum of the disorder, genotype–phenotype correlations, and the effect of different missense variants on CHD4 function.MethodsWe collected clinical and molecular data from 32 individuals with mostly de novo variants in CHD4, identified through next-generation sequencing. We performed adenosine triphosphate (ATP) hydrolysis and nucleosome remodeling assays on variants from five different CHD4 domains.ResultsThe majority of participants had global developmental delay, mild to moderate intellectual disability, brain anomalies, congenital heart defects, and dysmorphic features. Macrocephaly was a frequent but not universal finding. Additional common abnormalities included hypogonadism in males, skeletal and limb anomalies, hearing impairment, and ophthalmic abnormalities. The majority of variants were nontruncating and affected the SNF2-like region of the protein. We did not identify genotype–phenotype correlations based on the type or location of variants. Alterations in ATP hydrolysis and chromatin remodeling activities were observed in variants from different domains.ConclusionThe CHD4-related syndrome is a multisystemic neurodevelopmental disorder. Missense substitutions in different protein domains alter CHD4 function in a variant-specific manner, but result in a similar phenotype in humans.     

Genotype-phenotype associations in a large PTEN Hamartoma Tumor Syndrome (PHTS) patient cohort

BackgroundPathogenic PTEN germline variants cause PTEN Hamartoma Tumor Syndrome (PHTS), a rare disease with a variable genotype and phenotype. Knowledge about these spectra and genotype-phenotype associations could help diagnostics and potentially lead to personalized care. Therefore, we assessed the PHTS genotype and phenotype spectrum in a large cohort study.MethodsInformation was collected of 510 index patients with pathogenic or likely pathogenic (LP/P) PTEN variants (n = 467) or variants of uncertain significance. Genotype-phenotype associations were assessed using logistic regression analyses adjusted for sex and age.ResultsAt time of genetic testing, the majority of children (n = 229) had macrocephaly (81%) or developmental delay (DD, 61%), and about half of the adults (n = 238) had cancer (51%), macrocephaly (61%), or cutaneous pathology (49%). Across PTEN, 268 LP/P variants were identified, with exon 5 as hotspot. Missense variants (n = 161) were mainly located in the phosphatase domain (PD, 90%) and truncating variants (n = 306) across all domains. A trend towards 2 times more often truncating variants was observed in adults (OR = 2.3, 95%CI = 1.5–3.4) and patients with cutaneous pathology (OR = 1.6, 95%CI = 1.1–2.5) or benign thyroid pathology (OR = 2.0, 95%CI = 1.1–3.5), with trends up to 2–4 times more variants in PD. Whereas patients with DD (OR = 0.5, 95%CI = 0.3–0.9) or macrocephaly (OR = 0.6, 95%CI = 0.4–0.9) had about 2 times less often truncating variants compared to missense variants. In DD patients these missense variants were often located in domain C2.ConclusionThe PHTS phenotypic diversity may partly be explained by the PTEN variant coding effect and the combination of coding effect and domain. PHTS patients with early-onset disease often had missense variants, and those with later-onset disease often truncating variants.     

Congenital heart disease and aortic arch variants associated with mutation in PHOX2B

PurposeCongenital central hypoventilation syndrome (CCHS, OMIM 209880) is a rare autosomal dominant disorder caused by mutation in PHOX2B that manifests as a consequence of abnormal neural crest cell migration during embryogenesis. Unlike other neurocristopathies, however, its impact on the cardiovascular system has not been previously assessed. This study was an effort to characterize the association between congenital heart disease (CHD) and mutations in PHOX2B in patients with CCHS.MethodsA retrospective review of patients with CCHS in conjunction with functional analysis of PHOX2B mutations associated with CHD was performed. To substantiate functional implications of identified variants, we conducted protein structure analyses and in silico mutagenesis were conducted.ResultsThe prevalence of CHD among patients with CCHS was significantly greater (30%; p < 0.001) than that of the current estimated prevalence of CHD. The majority of patients had anomalies involving the proximal aortic arch and/or proximal coronary arteries. Variants associated with CHD in this cohort appear to disrupt DNA binding of PHOX2B via alteration of its homeobox domain.ConclusionThis is the first report of an association between CHD and mutation in PHOX2B. Results are highly suggestive that alteration or elimination of the homeobox domain conveys significant risk for associated CHD or aortic arch variation.